Fig. 6

Roquin1 bound to the stem–loop structure in the 3′UTR of cell cycle–promoting genes. a Schematic representation of the luciferase reporter constructs of CCNE1 and MCM2 containing truncated 3′UTRs without the stem–loop structure (Δ stem–loop). b Measurement of luciferase activity of reporters containing full-length 3′UTRs or truncated 3′UTRs (Δ stem–loop) of CCNE1 and MCM2, respectively. The results shown represent the mean ± standard deviation of four independent experiments. *P < 0.05. c Schematic representation of the luciferase reporter constructs of human β-actin 3′UTR containing the stem–loop structure of CCNE1 (w/CCNE1 stem–loop) or MCM2 (w/MCM2 stem–loop). d Measurement of luciferase activity of reporters containing wild-type β-actin 3′UTRs or β-actin 3′UTRs with stem-loop sequences of CCNE1 and MCM2. *P < 0.05. e The predicted stem–loop structures of CCNE1 (top) and MCM2 (bottom) in their 3′UTRs and mutation strategy (asterisks indicate base substitution). Mutant1 was unable to form a stem–loop structure (middle), and Mutant2 still formed a stem–loop structure (left). f Luciferase assays were conducted using reporters from (E) along with Roquin1 or control vector, and then measurement of the luciferase activity. *P < 0.05. g Roquin1/GFP fusion protein was expressed in MDA-MB-468 cells for 24 h, and then cell lysates were collected. Biotinylated CCNE1 and MCM2 wild-type or mutant probes (mut1 and mut2) were used for the pull-down assay. h RIP-ChIP assay was conducted with genome fragments from MDA-MB-468 cells after Roquin1/GFP fusion protein expression for 24 h